Infection by human immunodeficiency virus (HIV) in vivo often involves a long latency period before the development of an acute virally-mediated disease (e.g., AIDS). The events that trigger a transition from latent viral infection to full-blown AIDS are not completely understood (See, e.g., Paul (ad) Fundamental Immunology Third Edition, Raven Press Ltd., New York (1993) Chapter 39 for an overview of HIV infection and AIDS). However, the activation of the HIV-LTR promoter is dependent upon factors that are normally involved in regulating lymphokine production necessary for T cell activation. In many respects, HIV-1 LTR regulation is similar to, for example, the regulation of IL-2 (see Siekevitz et al., Science (1987) 238 (4833): 1575-1578; Shaw, et al. (1988), Science 241: 202-205; Masuda et al. (1993) Molecular and Cellular Biology 13(12): 7399-7407; Chuvpilo et al. (1993) Nuc. Acids Res. 21(24): 5694-5704, and Lowenthal (1988) Proc. Natl. Acad. Sci. U.S.A. 85:4468-4472).
It is the similarity between HIV-LTR activation and T cell activation that is thought to account for the seemingly paradoxical effect of cyclosporin A upon HIV induction. Cyclosporin A is a known T cell suppressive agent, so it would not appear to be a likely candidate as an AIDS therapeutic, since AIDS also results in T cell suppression. However, the similarity between T cell activation mechanisms and HIV-activation mechanisms actually suggested that cyclosporin A would act to inhibit the HIV LTR, since it was known to inhibit T cell activation.
Siekevitz et al. (supra) demonstrated that cyclosporin A inhibited T cell specific mitogenic lectin induction of the LTR promoter by phytohemagglutinin A (PHA). However, activation of the HIV LTR by other mitogenic agents such as the mitogenic phorbol ester phorbol 12-myristic 13-acetate (PMA) were not inhibited by cyclosporin A. Despite the apparently limited ability of cyclosporin A to inhibit the HIV LTR, the drug is now in clinical trials as a potential AIDS therapeutic agent.
PHA and PMA both affect a variety of T cell activities, as well as expression from the HIV-LTR. Each of the mitogens stimulates signal transduction pathways involved in T cell activation which are partly modulated through calcium-dependent mechanisms (see Alberts et al., Molecular Biology of The Cell second edition Garland Publishing, Inc New York and Paul (1989); Paul (ed) Fundamental Immunology Third Edition, Raven Press Ltd., New York (1993) and Cole et al., Cancer Metastasis Rev. 13(1): 31-44 (1994)). For instance, the action of the endogenous activator of the calcium-dependent protein kinase C, diacylglycerol, is mimicked by phorbol esters such as PMA which are known to induce expression by the LTR promoter (Siekevitz et al. supra). Diacylglycerol is produced by hydrolysis of membrane phospholipids, such as phosphatidylinositol bisphosphate. Phosphatidylinositol bisphosphate is hydrolyzed to diacylglycerol and inositol triphosphate by phospholipase C-.beta. (PLC-.beta.) and phospholipase C-.gamma. (PLC-.gamma.). These enzymes are regulated through different signal transduction pathways. PLC-.beta. is stimulated in response to ligand binding to transmembrane receptors which associate with guanine nucleotide binding protein intermediates (Berridge, et al., Nature 341:197-205 (1989)).
PLC-.gamma. is stimulated by specific tyrosine phosphorylation by a receptor tyrosine kinase stimulated by ligand binding (Aaronson, Science 254:1146-1153 (1991)). This process is dependent upon either calcium influx or intracellular calcium mobilization (Gusovsky, et al., J. Biol. Chem. 268:7768-7772 (1993); Tanaguchi, et al., J. Biol. Chem. 268:2277-2279 (1993); and Chapron, et al., Biochem. Biophs. Res. Comm. 158:527-533 (1989)). The activated PLC-.gamma. hydrolyzes membrane phosphatidyl inositol bisphosphate to yield diacylglycerol and inositol polyphosphates, which act as second messengers. Diacylglycerol may be involved in vivo with activation of HIV as suggested by phorbol ester-induction experiments, or it may be the upstream activator of other signaling molecules which operate through mobilization of calcium. PHA binds to receptor molecules, e.g., with the carbohydrate structure Gal.sup..beta.1,4 GlcNAc.sup..beta.1,6 Man.sup..beta.1,2 GlcNAc.sup..beta.1,4 Gal (Stryer, Biochemistry Third Edition (1989) ISBN 0-7167-1843-X p. 345). PHA is also thought to activate gene expression secondary messenger systems that involve Calcium-mediated signaling events, although the details of signal transduction are not presently known (Paul, supra).
Compound 1 (carboxyamidotriazole or "CAI"), shown below, is a calcium response modifier with antiproliferative and antimetastatic activities (Kohn, et al., J. Natl. Cancer Inst. 82:54-60 (1990); Felder, et al., J. Pharm. Exp. Therapeut. 257:967-971 (1991); Kohn, et al., Cancer Res. 52:3208-3212 (1992) and Kohn et al U.S. Pat. No. 5,132,315 (1992)). Compound 1 inhibits receptor-operated and voltage-gated calcium influx (Felder, et at, J. Pharm. Exp. Therapeut. 257:967-971 (1991); Hupe, et al., J. Biol. Chem. 266:10136-10142 (1991)), calcium-dependent arachidonic acid release (Felder, et al., J. Pharm. Exp. Therapeut. 257:967-971 (1991); Clark, et al., Cell. 65:1043-1051 (1991)), and tyrosine kinase phosphorylation and concomitant activation of phospholipase C-.gamma. (Gusovsky, et al., J. Biol. Chem. 268:7768-7772 (1993)). The ability of CAI to inhibit selected calcium-mediated signal transduction events made it an ideal tool with which to investigate the role of calcium regulation underlying the activation of the HIV LTR promoter. ##STR1##
The present invention demonstrates that CAI inhibits activation of the HIV LTR promoter by the phorbol ester 12-myristic 13-acetate (PMA) and the mitogenic lectin phytohemagglutinin (PHA). Thus, the inhibition of the HIV LTR which is provided by CAI is more general than that provided by cyclosporin A, and in all likelihood represents inhibition of the signal transduction events leading to LTR activation at a different point in the regulatory cascade than cyclosporin A. This discovery provides for the use of calcium-flux modulators as AIDS therapeutics, as well as for a variety of improvements to in vitro manipulations of HIV-infected cells. These calcium-flux modulators can be compounds which are structurally related to CAI, and which are known to have similar biological properties, such as those described in co-pending application Ser. No. 08/209,089, or they can be chemically unrelated compounds that are known to have similar biological properties. For instance, Compound 2 (below) is not closely related to CAI structurally, but it is known to have comparable effects on receptor-operated calcium influx (See, Gusovsky, et al., J. Biol. Chem. 268:7768-7772 (1993) and Merritt, et al., J. Biol. Chem 271:515-522 (1990)), and can be used to inhibit the HIV-LTR. ##STR2##